Draft Genome Sequence of an Extreme Haloarchaeon 3A1-DGR Isolated from a Saltern Crystallizer of the Little Rann of Kutch, India

Abstract

Haloarchaea are predominant in the salt crystallizers of the Rann of Kutch when the concentration of salts approaches saturation levels. The obligate and extreme halophilic archaeon 3A1-DGR, isolated from a salt crystallizer pond of the Little Rann of Kutch, India, needs minimum of 10 % NaCl in the growth medium. To understand the mechanism(s) of osmotolerance and adaptation at extreme osmolarity, and to mine relevant gene(s), the genome of this haloarchaeon, 3A1-DGR, was sequenced. We report here, the 2.88 Mb draft genome sequence of the haloarchaeon 3A1-DGR, with G+C content of 68 % and the possible involvement of 43 genes in stress tolerance. Further studies of the genome of this haloarchaeon would be required to identify gene(s) that might be responsible for imparting extreme osmotolerance.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0483-7) contains supplementary material, which is available to authorized users.

Archaea are believed to have evolved 4 billion years ago [1], and can survive in the extremities of pH, temperature, pressure, salts, etc., besides playing significant roles in the bio-geochemical cycles [2]. The salt marshes of the Great and the Little Rann of Kutch of Gujarat, India, harbour extreme halophilic archaea, besides few genera of bacilli like Bacillus, Salinibacillus, Thalassobacillus, Sediminibacillus, etc. The concentration of salts in these hypersaline regions and that of the salt crystallizers gradually approaches the point of saturation when salt starts crystallizing, allowing specific group of organisms to thrive in such conditions. The genomes of a number of bacilli of the genera Bacillus, Salinibacillus, Thalassobacillus, and Sediminibacillus, isolated from the salt crystallizers of the Little and Great Rann of Kutch, have been sequenced recently to understand the mechanism(s) of halophilism(s) and to isolate relevant gene(s) [3–7].

The present haloarchaeon 3A1-DGR was isolated from a sample (W3A; total salts concentration of 418.7 g/l; pH: 6.7) collected from a salt crystallizer pond (N 23°44.30′ E 71°10.81′) of the Little Rann of Kutch, Gujarat, India. This extreme halophilic archaeon was isolated by spread plating of the diluted samples into different known standard halophilic media including complex medium (CM), minimum growth medium (MGM), modified growth medium for haloarchaea, halophilic medium (HM), standard growth medium (SGM), DSMZ medium 1184, DSMZ medium 97, etc. containing different concentrations of NaCl (18–23 %) [8–11]. The Petri dishes were incubated at different temperatures (28, 37 and 42 ^°C) for 15–20 days.

The haloarchaeon 3A1-DGR is an isolate of a lineage of isolates from the salt crystallizers of the Little Rann of Kutch, India and has been described recently [12]; which lies between Natronomonas and Halopenitus on the basis of partial 16S rRNA sequences. The lineage consists of three haloarchaeal isolates, namely 3A1-DGR (16S rRNA GenBank acccession no. JF802160), H9-DGR, and 2ANA-DGR.

Comparison of 3A1-DGR with Halopenitus revealed that whereas Halopenitus persicus DC30(T) is pleomorphic (rod to triangular or disc shaped), forms pale pink-pigmented colonies, needs at least 0.02 M MgCl₂ for growth, optimum being 0.1 M [13], the cells of 3A1-DGR are irregularly coccoid (outer diameter of 1.4–1.5 µm) and formed hard and tough colonies which produce creamy pigmentation when incubated in dark, and light red pigmentation in illuminated conditions [12], and does not require any MgCl₂ for growth. Moreover, G + C content of 3A1-DGR is 68 % and contains phosphotidylglycerol phosphate (PGP), phosphotidyl glycerol phosphate methyl ester (PGPME) and diglycosyl diether (DGD) as major polar lipids against G+C content of 66 % and major polar lipids of PGP and PGPME in Halopenitus persicus DC30(T). It also differs from Halopenitus malekzadehii CC65 (T) by the fact that CC65(T) needs at least 0.02 M MgCl₂ for initiation of growth, 0.4 M being optimum; produces light yellow colonies and has G+C content of 63.8 % [14]. Studies revealed that the obligate haloarchaeon 3A1-DGR needs a minimum of 10 % NaCl in growth media for initiation of growth and can continue to grow up to 35 % NaCl and 42 °C, with optimum growth requirement of around 20 % NaCl, 40 °C and pH 7.5 at which it can reach a log phase of growth in around 5 days (Fig. 1) in complex medium (CM). The present isolate does not survive lyophilisation because of obligate halophilic nature and hence preserved in glycerol stock at −80 °C; in stabs and in slants at 4 °C, and in liquid broth at room temperature keeping minimum of 10 % of NaCl in the growth media and sub-cultured bi-monthly for maintenance and further use.

Fig. 1.

Growth curve of extreme haloarchaeon 3A1-DGR at 20 % NaCl in CM medium

To understand the mechanisms of obligate halophilism, the genome of 3A1-DGR was sequenced by Roche 454 genome analyser (GS FLX). Both shotgun and 3 KB mate-paired library sequencing were performed. In shotgun sequencing, 842518 reads with average read length of 468 bases were obtained. However, in mate-paired (3 KB) library sequencing, 153960 and 106333 reads, respectively, with average read lengths of 455 and 423 bases, respectively, were obtained.

The genome was assembled de novo by GS de novo assembler v2.9 [15] with genome coverage of about 173X. The draft assembly of 3A1-DGR resulted into 3 scaffolds of 2880902 bases with the largest scaffold of 1817982 bases. The N₅₀ scaffold length of 1817982 bases with an average size of 960300 bases (minimum 5278 bases; maximum 1817982 bases) was achieved. The assembly consists of 15 contigs (average size 192033 bases) with the N₅₀ contig of 425867 bases and largest contigs of 628022 bases. All the assembly data were deposited in the DDBJ/EMBL/GenBank nucleotide sequence database.

The draft genome of 3A1-DGR was annotated further using Rapid Annotation of Subsystem Technology (RAST) server [16], Glimmer 3 [17, 18], GeneMarkS [19], tRNAScan-SE [20], RNAmmer [21], KEGG [22], and Signal P [23] for predicting subsystems, coding sequences (CDS), tRNA, rRNA genes, signal peptides, biochemical pathways, etc.

Using the different software tools, we identified 2894 coding sequences (CDS). The draft genome also encodes 46 RNA genes (43 tRNA and 3 rRNA genes) and 254 subsystems. There was one each of 5S, 16S and 23S rRNA in the draft genome of the haloarchaeon 3A1-DGR. Among the CDS, 1931 are not in a subsystem (nonhypothetical, 540; hypothetical, 1391), whereas 963 (nonhypothetical, 936; hypothetical, 27) are in a subsystem. This indicates that nearly 66 % of the CDS could not be placed in a subsystem and nearly 49 % of the CDS have hypothetical functions. RAST annotation also revealed the association of 43 genes in stress responses in this organism: 10 in osmotic stress (10 in choline and betaine uptake and betaine biosynthesis), 28 in oxidative stress (2 in protection from reactive oxygen species [ROS], 16 in oxidative stress, 2 in glutathione:biosynthesis and gamma-glutamyl cycle, 4 in glutathione:non-redox reactions, 3 in rubrerythrin, and 1 in glutaredoxins), 2 in detoxification and 3 in no subcategory. Further analysis identified 15 signal peptides, and three closest neighbours of haloarchaeon 3A1-DGR as Halogeometricum borinquense DSM 11551 (genome ID 469382.4), Haloquadratum walsbyi DSM 16790 (genome ID 362976.10) and Haloarcula marismortui ATCC 43049 (genome ID 272569.1) with <90 % similarity. In addition, 306 genes have been mapped to different pathways involved in the biosynthesis and degradation of amino acids and derivatives, including 50 in branched-chain amino acid pathways. Similarly, 167 genes have also been mapped to different pathways of central carbohydrate metabolism including 26 in the serine-glyoxylate cycle.

To unravel the mechanism(s) of osmotolerance and to identify relevant gene(s), comparative genomics will be initiated on completion of the filling of the gaps in the sequence data. Deciphering the functions of hypothetical proteins would also lead to identification of a number of novel genes. Future studies are underway in this direction.

Nucleotide sequence accession numbers

This Whole Genome Shotgun (WGS) project has been deposited at DDBJ/EMBL/GenBank under the accession number ATCR00000000. The version described in this paper is version ATCR02000000. Bioproject registered under accession: PRJNA183189 ID:146531.